Isolation and characterization of eight
pregnancy-associated glycoproteins present
at high levels in the ovine placenta
between day 60 and day 100 of gestation

a
Physiology of Reproduction, Faculty of Veterinary Medicine, University of Liege, 4000, Belgiumb
INRA, CRRA de Meknès, BP 578, Morocco

(Received 23 September 2003; accepted 2 February 2004)

Abstract - Pregnancy-associated glycoproteins (PAG), structurally related to aspartic proteinases, are expressed in the outer epithelial
cell layer (chorion/trophectoderm) of the ungulate placenta. The aim of the present study was to isolate as many PAG molecules
as possible from placentae collected between day 60 and day 100 of gestation and to characterize their amino-terminal amino-acid
sequences. Three heterologous radioimmunoassays were used to monitor PAG immunoreactivity throughout the isolation procedures.
Sequential use of DEAE-cellulose, gel filtration, and CM ceramic chromatographies led to the isolation of several fractions
rich in PAG immunoreactivity. The fractions with a large amount of proteins were also purified by chromatofocusing. The analysis
of immunoreactive fractions by SDS-PAGE, Western blotting and two-dimensional electrophoresis followed by amino-terminal microsequencing
on PVDF membranes allowed to identify eight different ovPAG with apparent molecular masses ranging from 55 to 66 kDa and isoelectric
points from 4.0 to 6.8. The N-terminal sequences were determined and their comparison to those previously identified revealed
that four of them are identical to those encoded by previously known cDNA, while the additional four sequences appear to be
novel since they have not yet been described.